Procedure for Bradford Assay

BSA from Signma Aldrich® was used to make a stock solution of 1mg/mL. BSA stock solution was made the following way:

10mg of BSA was weighted out

10mg of BSA was poured into a 10mL volumetric flask

10mL of sterile water was added to the 10ml volumetric flask

BSA and water were mixed until dissolved

6X Bradford reagent from Bio Rad® was diluted 6 fold to make 1X Bradford reagent. 1X Bradford was diluted by mixing 4mL of 6X Bradford reagent with 20mL of sterile water. 1X Bradford reagent was used to make Bradford assay standard solutions.

Standard solutions were made with different amounts of 1mg/mL BSA, 1X bradford reagent, and water. All sample solutions made up a total volume of 1.7mL to place in plastic cuvette. The volume of each reagent used are listed in table below:

BSA added[mL]

1X Bradford Reagent added[mL]

Sterile Water added[mL]

0

1.4

0.03

0.005

1.4

0.025

0.01

1.4

0.02

0.015

1.4

0.015

0.02

1.4

0.01

0.025

1.4

0.005

0.03

1.4

0

Each samples in plastic cuvette were placed in UV-vis spectrometer under spectrum method with wavelength ranging from 500nm to 800nm. The absorbance was recorded for analysis.

After the Bradford assay were run, 7.5μL of ADA protein fractions were mixed with 715μL 1X Bradford assay and 7.5μL water to test absorbance at 595nm.

Results and Calculations of ADA protein Concentrations

The calculated final BSA concentration is shown in table below:

Volume BSA Stock Added [mL]

VolumeTotal [mL]

[BSA] [mg/mL]

0

1.7

0

0.005

1.7

0.003566434

0.01

1.7

0.007132867

0.015

1.7

0.010699301

0.02

1.7

0.014265734

0.025

1.7

0.017832168

0.03

1.7

0.021398601

Absorbance at 595nm were plotted on a graph against the final concentration of BSA used before putting into UV-vis spectrometer. The graph is shown below:

Best fit line was shown in graph as function to calculate ADA protein concentration once absorbance was measured. The absorbance for ADA protein was kept below 1 to make sure measurement was within the limit of Bradford assay.

With the calculated best fit line, the absorbance of each protein fraction was used to calculate ADA protein concentration by fitting into the equation:

Absorbance = 27.609 * [BSA]mg/mL + 0.4363

Table including the absorbance obtained and final calculated ADA protein concentration in molarity is listed below:

-

Absorbance []

Conc. ADA [mg/mL]

Mass of ADA [mg]

Moles of ADA [mol]

Molar Absorptivity of ADA [mL/mg cm]

Molarity [M]

Fraction Oct

0.784

0.012459254

0.017816733

4.38E-10

62.92511628

5.84E-05

Fraction 2

0.559

0.004310033

0.006163347

1.51E-10

129.697395

4.66E-05

Fraction 1&3

0.501

0.002209344

0.003159363

7.76E-11

226.7640984

1.04E-05

Thus, the final concentration of ADA protein fraction made on 10/03/2012 is 58.4mM. Final concentration of ADA protein fraction 2 made on 11/06/2012 is 46.6mM. And final concentration of ADA protein fraction 1&3 made on the same day is 10.4mM.

Procedure for Making Au/HRP samples

Au/HRP samples were made with the mole ratios listed below:

10 - 50 - 100 - 150 - 200 - 250 - 300 - 350 - 400 - 450

28.5uM HRP stock solution was made. HRP from Alfa Aesar® has a molecular weight of 40,000 g/mol. 0.0057g of HRP was weighted out and mixed with 5mL of distilled water. The following calculations was done to obtain masses weighted for 28.5uM HRP stock solution:

HAuCl4 used for making Au/HRP samples were made on 09/05/2012 and has a concentration of 10.5mM.

Volume of HRP was set at 280.7uL while volumes of HAuCl4 were ranging from 7.62μL to 342.8μL. Distilled water was added until volume of sample at each mole ratio reached 8mL. Detailed volumes for each reactant is listed in table below:

Au/HRP ratio

HRP added [uL]

HAuCl4 added [uL]

Water added [uL]

Final concentration of Au[uM]

Final concentration of HRP[uM]

10

280.7

7.62

7711.68

1

10

50

280.7

38.1

7681.2

1

50

100

280.7

76.2

7643.1

1

100

150

280.7

114.3

7605

1

150

200

280.7

152.4

7566.9

1

200

250

280.7

190.5

7528.8

1

250

300

280.7

228.5

7490.8

1

300

350

280.7

266.6

7452.7

1

350

400

280.7

304.7

7414.6

1

400

450

280.7

342.8

7376.5

1

450

Each Au/HRP samples were capped, wrapped with aluminum foil, and placed in incubator at 85°C for 4 hours then cooled down to room temperature.